More than 1.5 million of the American women alive today have been or will be diagnosed with premature ovarian failure (POF) during their lifetimes and an unknown, probably much larger number will have early menopause without meeting the diagnostic criteria for POF. POF is characterized by accelerated depletion of ovarian follicles and decreased oocyte quality, but the causes remain unknown in 90% of cases. Polycyclic aromatic hydrocarbons (PAHs) are formed by the incomplete combustion of organic materials. Women are ubiquitously exposed to benzo[a]pyrene (BaP) and other PAHs via food, air pollution, and tobacco smoke. BaP is a potent ovotoxicant, and the developing ovary is particularly sensitive. Exposure to tobacco smoke, which contains high concentrations of BaP and other PAHs, is associated with decreased fecundity and earlier menopause in the daughters of women who smoked during pregnancy. We have shown that prenatal exposure of mice to BaP during primordial germ cell migration through the onset of meiosis causes POF in the F1 female offspring at doses that do not affect ovarian follicle numbers in the mothers. We further showed that embryos deficient in synthesis of the antioxidant glutathione (GSH) due to deficiency in the modifier subunit of glutamate cysteine ligase (Gclm) are more sensitive to the transplacental ovotoxicity of BaP than wild type littermates. Our preliminary data further show that BaP induces apoptosis in germ cells of cultured fetal ovaries and that Gclm null ovaries are more sensitive to the induction of germ cell apoptosis by BaP. In the current proposal we will test the hypothesis that BaP depletes germ cells in the prenatal ovary by inducing oxidative stress and apoptosis, while inducing heritable epigenetic changes in surviving germ cells to cause accelerated depletion of ovarian follicles in subsequent generations, and that GSH is protective against these effects. We will test this hypothesis in two aims: 1) To establish the critical window of development for and mechanisms of the transplacental ovotoxicity of BaP and to define the mechanism by which GSH modulates BaP-induced ovotoxicity during the critical window. We will use complementary in vivo transplacental exposure and cultured embryonic ovary models. We will test the potential protective effects of supplementation with GSH and other antioxidants. 2) Test whether the ovarian phenotype of prenatal exposure to BaP is transgenerational and is mediated by epigenetic changes in the germ line. We will utilize RNA- sequencing to examine genomewide gene expression and will assess global DNA methylation using MBD- Sequencing in F1, F2, and F3 primordial germ cells from BaP exposed compared to control lineages.
Early menopause due to early cessation of ovarian function affects more than one percent of all women, but the causes are largely unknown. The project will uncover the mechanisms by which prenatal exposure to the widespread pollutants PAHs causes premature ovarian failure in daughters, as well as potentially in granddaughters and great-granddaughters of exposed females. The project will determine the most sensitive time during prenatal development for these effects and how deficiency in the antioxidant glutathione increases sensitivity during these sensitive windows, paving the way for prevention of adverse ovarian effects of PAHs.
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